The Adeno-associated viruses (AAVs) are ssDNA packaging viruses belonging to the Dependoparvovirus genus of the Parvoviridae. Gene delivery systems based on the AAVs recently entered an exciting phase with the FDA approval of Luxturna, an AAV serotype 2 (AAV2)-based gene therapy for treating a monogenetic defect in the eye. However, the success of Luxturna was ushered by the fact that the eye is an immune privileged organ and direct administration avoids pre-existing host immunity. This remains a significant challenge to the therapeutic efficacy of the AAV gene delivery system. More recently, members of the Bocaparvovirus genus of the Parvoviridae have also been developed as viral gene delivery vectors, also for treating monogenetic diseases. However, high level of seroprevalence of host antibodies against AAVs and bocaviruses (BoVs), at ?70%, represents a major challenge to full therapeutic realization of both systems. The primary focus of this project has been to characterize the antigenic structures of primate AAVs, using mouse monoclonal antibodies (Mabs), as they relate to capsid determinants of receptor attachment, tissue tropism, and transduction efficiency (gene expression). We pioneered the use of this information for molecular engineering of AAV vectors able to escape antibody recognition and currently under evaluation as potential clinical vectors. However, there is need to confirm that the ?polyclonal? information obtained by mapping several mouse Mabs for each AAV serotype studied recapitulates the polyclonal human response. In this renewal application, we will characterize the ability of human and non-human primate (NHP) sera to neutralize or bind and not neutralize vector transduction. This will guide the engineering of antibody escape and/or transduction efficacy and thus therapeutic utility. We expand our viral models to include non-primate AAVs and BoV vectors in an effort to expand the pool of parvoviral vectors available for use. Our three specific aims will ask four new questions: (1) ?Do primate antibodies share epitopes with the previously described murine Mabs?? (2) ?Do the binding sites of neutralizing and non- neutralizing binding antibodies overlap?? (3) ?Do non-primate AAVs naturally escape pre-existing neutralizing primate antibodies and capable of transducing human cells?? And (4) ?Can we engineer the antigenic sites on BoV vectors to evade neutralization by antibodies while retaining or improving the parental transduction efficiency??. We will use cryo-electron microscopy and image reconstruction to determine high-resolution structures of non-primate AAVs and BoV capsids, alone and in complex with glycan receptors, to ?3 resolution and the structures of AAV/BoV capsid ? human/NHP antibodies to between 3 to 4 resolution. This is routine in our group. We will use the information obtained to engineer vectors that retain their cell binding properties but evade recognition by human/NHP. We will evaluate these vectors in vitro and in vivo in the presence of IgG and IVIG, respectively. We will create new clinical biologics, as was done in the past funding round, to expand the parvovirus viral vector repertoire, thus the number of treatable diseases targets, and cohort of treatable patients.